Many insect species exert a negative impact on human health through serving as disease propagation vectors or through causing agricultural damage. Chemosensory receptors, odorant receptors (ORs) and taste receptors, are critical to the ability of insects to function in these roles. Insect chemosensory receptors are an entirely novel receptor class, having no genetic or structural relationship to any known receptor family (including mammalian chemosensory receptors). Thus, these receptors are appealing targets for the development of new compounds for insect control. Realizing this goal requires development of a detailed understanding of insect chemosensory receptor structure and function. However, one of the most notable characteristics of these receptors is how little we know about their structure. In this project, we will identify structural features of insect ORs that are important in odorant binding and recognition. We have established a robust functional assay for insect odorant receptors using the Xenopus oocyte expression system and robotic electrophysiology. Using this assay, we are proposing a project that will determine the structural basis for odorant recognition by insect ORs. In Aim 1, we will express and screen a large panel of Drosophila melanogaster odorant receptors (DmORs) using our assay. In Aim 2, we will identify regions of interest on the odorant binding subunits of DmOrs by screening the receptor panel with cysteine-reactive methanethiosulfonate reagents. This screen will take advantage of the natural distribution of cysteine residues across the DmOR family. In Aim 3, we will use the Substituted Cysteine Accessibility Method (SCAM) to explore critical functional regions (identified in Aim 2) of the odorant binding subunits of insect ORs. In Aim 4, we will use site-directed mutagenesis and functional analysis to reveal the structural basis for odorant recognition by insect odorant receptors. This project will provide an understanding of odorant ligand recognition by insect ORs at the molecular level. This project is important to human health because insect ORs are a novel target for the development of compounds to control deleterious insect species.